Despite over 60 years of successful chemotherapy, Tuberculosis remains the leading cause of human mortality due to an infectious bacteria. The antibiotics used in current short course treatment (6 months) are atypical and with the exception of rifampicin, are uniquely used in the treatment of human Tuberculosis. Despite this, clinical resistance to the extant antitubercular compounds is rising rapidly, with multi-drug resistant isolates comprising >6% of recent isolates. More threatening are the recent reports of pan-resistant TB isolates in South Africa which are resistant to 16 current first, second and third line antituberculars. Over the last fifteen years, this program has sought to identify essential bacterial biosynthetic pathways that are present in TB, but not the human host;including the biosynthetic pathways of L-lysine, pantothenate, L-leucine and L-arginine. In this proposal we will use a similar integrated combination of kinetic, mechanistic, isotopic and structural approaches to define the structures and mechanisms of enzymes involved in the biosynthesis of mycothiol, a small molecular weight thiol present in mycobacterial species, that serves a physiological function similar to that of glutathione in the host. Specifically we will focus on the mshA- and mshC-encoded enzymes that catalyze the formation of the mycothiol pseudodisaccharide and the cysteine ligase, respectively. In a separate series of experiments we will determine the structure and function of a selected number of enzymes that we have recently shown are capable of tightly binding the isonicotinoyl adducts of NAD and NADP that are the bacteriostatic forms of the prodrug isoniazid. Specifically, we will focus on the ribG-encoded bifunctional deaminase-reductase that is the second step in the biosynthesis of riboflavin. Finally, we will initiate studies on the M. tuberculosis blaC-encoded beta-lactamase. The long term goals of the present proposal are to develop these critical enzymes into targets for the design, or discovery, of novel new classes of compounds that will be starting points for the arduous process of converting inhibitors into effective antitubercular drugs. Tuberculosis continues to kill >1.6 million people every year. With the emergence and spread of multi-drug resistant strains of the bacteria, new targets must be identified and developed to which new inhibitors can be identified, allowing new therapeutically useful drugs to be developed.